GCB Bioenergy (2016), doi: 10.1111/gcbb.12342
Can miscanthus C4 photosynthesis compete with
festulolium C3 photosynthesis in a temperate climate?
XIURONG JIAO1, KIRSTEN KØRUP1, MATHIAS NEUMANN ANDERSEN1,
E R I K J . S A C K S 2 , X I N - G U A N G Z H U 3 , P O U L E R I K L Æ R K E 1 and U F F E J Ø R G E N S E N 1
1
Department of Agroecology, Aarhus University, Blichers Alle 20, DK-8830 Tjele, Denmark, 2Department of Crop Sciences,
University of Illinois, 1201 W. Gregory Dr., Urbana, IL 61801, USA, 3CAS-MPG Partner Institute for Computational Biology,
and Institute of Plant Physiology and Ecology, SIBS, Shanghai 200031, China
Abstract
Miscanthus, a perennial grass with C4 photosynthesis, is regarded as a promising energy crop due to its high
biomass productivity. Compared with other C4 species, most miscanthus genotypes have high cold tolerances at
14 °C. However, in temperate climates, temperatures below 14 °C are common and our aim was to elucidate
cold tolerances of different miscanthus genotypes and compare with a C3 perennial grass – festulolium. Eleven
genotypes of M. sacchariflorus, M. sinensis, M. tinctorius, M. 9 giganteus as well as festulolium were grown under
warm (24/20 °C, day/night) and three under cold (14/10 °C, 10/8 °C and 6/4 °C) conditions in a controlled
environment. Measurements of photosynthetic light response curves, operating quantum yield of photosystem II
(ΦPSII), net photosynthetic rate at a PAR of 1000 lmol m2 s1 (A1000) and dark-adapted chlorophyll fluorescence (Fv/Fm) were made at each temperature. In addition, temperature response curves were measured after
the plants had been grown at 6/4 °C. The results showed that two tetraploid M. sacchariflorus and the standard
triploid M. 9 giganteus cv. Hornum retained a significantly higher photosynthetic capacity than other miscanthus genotypes at each temperature level and still maintained photosynthesis after growing for a longer period
at 6/4 °C. Only two of five measured miscanthus genotypes increased photosynthesis immediately after the
temperature was raised again. The photosynthetic capacity of festulolium was significantly higher at 10/8 °C
and 6/4 °C than of miscanthus genotypes. This indicates that festulolium may be more productive than the currently investigated miscanthus genotypes in cool, maritime climates. Within miscanthus, only one M. sacchariflorus genotype exhibited the same photosynthetic capacity as Hornum at both cold conditions and when the
temperature was raised again. Therefore, this genotype could be useful for breeding new varieties with an
improved cold tolerance vis-a-vis Hornum, and be valuable in broadening the genetic diversity of miscanthus
for more widespread cultivation in temperate climates.
Keywords: C3 photosynthesis, C4 photosynthesis, chlorophyll fluorescence, cold tolerance, genotype difference, light response
curves, quantum yield, temperature response curves
Received 31 October 2015; accepted 12 December 2015
Introduction
Bioenergy crops are a potential replacement source for
fossil fuels. To make this option economically competitive and sustainable, major research issues include identifying crops that have a maximum biomass production
and at the same time a minimum environmental impact
when grown on marginal land under various abiotic
stress conditions (Gabrielle et al., 2014). Even though
the yield of crops depends on light interception, conversion and partitioning efficiencies, an extension of the
growing season has a significant positive impact on
biomass production (Zhu et al., 2010). To provide
Correspondence: Xiurong Jiao, tel. +45 8715 4761,
e-mail: [email protected]
sustainable biomasses/feedstocks in temperate climates,
the ideal bioenergy crop should be perennial and have
a high cold-tolerance to support a long growing season
in which photosynthesis can take place, thereby promoting high annual yields and low losses to the environment (Karp & Shield, 2008).
An important basic physiological trait influencing
cold sensitivity and productivity in crops is the difference between C3 and C4 photosynthesis. Photosynthetic
activity in C3 and C4 species differs under cold conditions. For C3 plants, the Rubisco capacity has been
regarded as the predominant constraint to photosynthesis under a wide range of temperatures at CO2 levels
below the current ambient concentration (Farquhar
et al., 1980; Sage & Kubien, 2007). The C4 photosynthesis
is usually advantageous at low CO2 concentrations and
© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
1
2 X . J I A O et al.
high temperatures, and thus its distribution is usually
limited at daily mean minimum temperatures of 8 to
10 °C or below during the period of active growth
(Teeri & Stowe, 1976; Long, 1983). However, it has been
found that the C4 Miscanthus 9 giganteus is capable of
reaching high photosynthetic efficiency even in October
with an average temperature of 10 °C in southern
England (Beale et al., 1996), and it is hypothesized that
the C4 sensitivity to low temperatures may not be a
generic and unbreakable trait (Long & Spence, 2013).
The perennial grass crop, miscanthus, has therefore
been regarded as one of the most ideal energy crops due
to its high biomass potential and low fertilizer requirements (Lewandowski et al., 2000) and because it can be
harvested yearly with high average yields ranging from
13.1 to 14.4 Mg ha1 for a period of more than 15 years
even in the cold temperate maritime climate of Denmark
(Larsen et al., 2014). Another productive perennial grass
under temperate conditions, but with the C3 photosynthetic pathway, is festulolium, which has been found
useful for extending the autumn grazing season in continental conditions (Skladanka et al., 2010). Festulolium is
a cross between species from the genus Festuca, with a
high level of general stress tolerance towards e.g.
drought and heavy grazing, and species from the genus
Lolium, which is the predominant forage grass in Europe
(Wilkins & Humphreys, 2003). Several festulolium cultivars, in which the good forage quality of ryegrass species has been combined with the high persistency and
stress tolerance of fescues, have been bred recently
(Østrem & Larsen, 2008; Halling, 2012). The cultivar festulolium cv. Hykor has been shown to have a high yield
potential of around 18 Mg ha1 and high persistency
throughout the season (www.DLF.com). Thus, festulolium may be ideal as a sustainable feedstock for biorefineries (e.g. protein feed and bioenergy) in temperate
climates (Parajuli et al., 2015).
In previous studies, 14 °C has been chosen as an
important temperature level for examining the cold tolerance of plants with C4 photosynthesis (Haldimann,
1996; Farage et al., 2006). We screened a range of miscanthus genotypes for their tolerance to a 14/10 °C
growing environment to find improved genotypes compared with M. 9 giganteus, but found only a few with
slightly improved characteristics (Jiao et al., 2016). However, plants in northern Europe are frequently exposed
to much lower temperatures, especially in the autumn
and spring seasons. Therefore screening of the photosynthetic response to temperatures below 14 °C may be
helpful to identify genotypes with a high tolerance to
low temperatures, which are critical for growth in the
spring and autumn in continental climates and throughout the growing season in cool temperate maritime
climates.
In a side-by-side trial in Illinois, Dohleman & Long
(2009) found that M. 9 giganteus was 59% more productive than maize (Zea mays) due to its larger leaf area and
its longer growing season that was facilitated by superior cold tolerance. Photosynthesis in M. 9 giganteus has
been found to still occur at 10 °C, although at a lower
rate than in plants grown at 14 °C (Farage et al., 2006).
Photosynthetic CO2 uptake of M. 9 giganteus and maize
measured during short-term exposure to 5 °C after
having been grown at 24 °C and 14 °C showed that M.
9 giganteus kept a photosynthetic level of around 4 to
5 lmol m2 s1, which was significantly higher than the
1 to 2 lmol m2 s1 of maize grown at 14 °C (Naidu
et al., 2003). However, it is unclear whether the photosynthetic apparatus in the miscanthus was damaged or
not and whether the photosynthetic capacity would
continue to function after long-term exposure to temperatures below 10 °C. The C3 photosynthesis of festulolium is expected to be tolerant to low temperatures,
but an exposure to 2 °C resulted in a dramatic decline
in maximum quantum yield of PSII (Fv/Fm), in
increased nonphotochemical quenching (NPQ) and in
reduced efficiency of energy conversion in the photochemical processes (qP) (Pociecha et al., 2010).
Previous studies on high-yielding and cold-tolerant
miscanthus have focused mainly on M. 9 giganteus cv.
Hornum (Larsen et al., 2014), although this genotype is
impossible to further improve by breeding since it is a
sterile triploid hybrid of a tetraploid M. sacchariflorus
and diploid M. sinensis (Greef & Deuter, 1993) with little
genetic diversity (Hodkinson et al., 2002; Clark et al.,
2014). However, M. sinensis and M. sacchariflous could
be used to breed new M. 9 giganteus cultivars for
broadening the genetic base which would be useful in
the breeding of high-yielding genotypes with improved
cold-tolerance or for growing on a wider scale (CliftonBrown & Lewandowski, 2002; Jorgensen, 2011). Other
miscanthus species may also contribute cold tolerance
to breeding, one possibility being M. tinctorius, which
often grows at high altitudes or latitudes.
Exploring the background for high cold-tolerance in
plants with C4 photosynthesis may be helpful before
introducing them in cool regions and broadening their
planting area. An earlier study has found that low-temperature photoinhibition associated with the xanthophyll cycle (zeaxanthin accumulated from violaxanthin
under high light conditions) is reversible and is a strategy for C4 species to tolerate cool climates, even though
it was more detrimental to carbon gain in the C4 grass
Muhlenbergia glomerata than in the C3 species Calamogrostis canadensis grown at 14/10 °C (Kubien & Sage,
2004a). Photo damage is characterized by a reduced
photosynthetic capacity, which cannot be redeemed by
a rising temperature. For example, low temperatures
© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12342
COLD TOLERANCE OF C4 VS. C3 PLANTS 3
affect both the light-harvesting apparatus and the
CO2-fixing enzymes of maize (Baker et al., 1983). A previous study has reported that the ability to sustain the
photosynthetic rate at a relatively high level at 14 °C in
some genotypes might be dependent on maintenance
and increase of Rubisco (Spence, 2012) and PPDK concentrations in leaves (Wang et al., 2008a,b).
C3 plants usually have a lower optimal temperature
and lower maximum photosynthetic rates, but have the
advantage of higher photosynthesis at low temperatures
than C4 plants (Pearcy et al., 1981; Yamori et al., 2014).
Still, it is unclear at which temperature level miscanthus
can compete with C3 species in cool climates, and to
what degree there is genetic variation within the genus
that may help improve the competitiveness of miscanthus. Increased knowledge of this will also be helpful for
guiding site selection and adapting the cultivation
practices for these two types of crop in cool climates.
The aim of this work was to examine the cold tolerance of different C4 miscanthus genotypes through
studies of net photosynthetic rate (An), PSII operating
efficiency (ΦPSII), maximum quantum yield (Fv/Fm)
and light response under various growth temperatures
down to 6 °C. Furthermore, the cold tolerance of C4
miscanthus was compared with one well-known,
highly-productive festulolium cultivar. Our hypothesis
was that we would be able to identify genotypes with a
higher photosynthetic performance under cold conditions than the M. 9 giganteus cv. Hornum and that these
genotypes may even compete with a C3 grass under the
variable, temperate, maritime conditions of northwestern Europe.
Materials and methods
Plant material
One C3 species, the grass festulolium (cultivar Hykor), which is
a hybrid of Lolium multiflorum and Festuca arundinacea (Cernoch
et al., 2004; Zwierzykowski, 2004; Kopecky et al., 2008), and 11
genotypes of the C4 genus Miscanthus representing four
different species (M. sinensis, M. sacchariflorus, M. tinctorius and
M. 9 giganteus) were selected for climate chamber experiments
(Table 1).
Table 1 Genotypes measured in the climate chamber experiments. The genotypes numbers 1 to 14 are from a previous climate
chamber experiment (Jiao et al., 2016); genotypes 15 to 19 are new material for this study
Identification
Identification in
previous publication
Species
Origin
Altitude (m)
Latitude (°N)
Ploidy
Tin-1
Sin-3
133/1*
56/1*
M. tinctorius
M. sinensis
350
280
36
36
2x¶¶
2x¶¶
900
600
36
36
EMI-5‡
EMI-1‡
M114¶
M. sacchariflorus
M. sacchariflorus
M. sacchariflorus
M. 9 giganteus
M. 9 giganteus
Ainukura, Honshu†
Kamiyoshino (5 km SE
of Kanazawa), Honshu†
Hakusan National park, Honshu†
South of Shirakawa, Honshu†
Japan
cv Hornum§, Larsen, Denmark
Tinplant, GmbH, Klein
Wanzleben, Germany**
South of Shirakawa, Honshu†
Japan†
Near highway M58, outside
of Birobidzhan turnoff, Russia
Near M60 North of Lermontavka
Bred in Czech Rep.‡‡
4x¶¶
4x¶¶
4x¶¶
3x‡
2x
600
36
67
49
86
47
Sac-10
Sac-11
Sac-12
Gig-13
Gig-14
M. sinensis
M. sacchariflorus
M. sacchariflorus
Sin-15
Sac-16
Sac-17
Sac-18
Fest-19
Hykor††
M. sacchariflorus
L. multiflorum 9
F. arundinacea
*Identification used in Glowacka et al. (2015b).
†Seed plants collected in Japan 1995 and grown in Denmark since 1996 (Kjeldsen et al., 1999).
‡Clones from ‘European Miscanthus Improvement’ project (Clifton-Brown et al., 2001).
§EMI-1 was equivalent to Hornum, due to extreme high genetic similarity (Glowacka et al., 2015a).
¶(Carrie et al., 2012).
**(Kim et al., 2012).
††Identification used in Cernoch et al. (2004); Østrem & Larsen (2008); Ambye-Jensen et al. (2013); Ostrem et al. (2013).
‡‡Acquired by DLF-Trifolium, Denmark (Zwierzykowski, 2004; Kopecky et al., 2008).
§§(Cernoch et al., 2004).
¶¶Ploidy levels were determined by flow cytometry described in Jiao et al. (2016).
© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12342
4x
2x¶¶
2x¶¶
6x§§
C O L D T O L E R A N C E O F C 4 V S . C 3 P L A N T S 11
photosynthetic apparatus (Farage et al., 2006). A previous study also found an increased ФPSII/ФCO2 ratio
for M. 9 giganteus grown at 10 °C compared with 24 °C
and 14 °C (Farage et al., 2006). Photoinhibitory damage
has been found to be a major challenge for C4 plants
grown at cold temperatures, for example, maize grown
at 17 °C or below (Long et al., 1983). It seems that M. 9
giganteus has the ability to minimize this damage by a
massive increase in zeaxanthin content to facilitate heat
dissipation in conjunction with induction of electron
transport to an acceptor other than CO2 (Farage et al.,
2006).
However, CO2 leakiness from the bundle sheath cells
back to mesophyll is an alternative energy expenditure
in the C4 photosynthetic pathway under high light conditions. Especially at low temperatures, where the
Rubisco capacity is an important limitation for sustaining C4 photosynthesis (Sage, 2002; Kubien et al., 2003),
the ΦPSII/ΦCO2 ratio will increase (Von Caemmerer,
2000). Increased values of ΦPSII /ΦCO2 at cool temperatures have also been observed in Muhlenbergia glomerata
measured at 10 and 5 °C by Kubien & Sage (2004b),
who concluded that leaking of CO2 through the bundle
sheaths was increased due to limited Rubisco capacity
(Rubisco content) at low temperatures. The significantly
higher ΦPSII /ΦCO2 ratio at 6/4 °C in our study might
indicate that Rubisco posed a major limitation to photosynthesis in all the miscanthus genotypes but not in
Fest-19. However, the underlying reasons for the
increase of ΦPSII /ΦCO2 were not determined in this
study.
Gig-13 and Sac-11 grown at 6/4 °C in the present
study showed much higher rates of photosynthesis at
all measured temperatures from 5 to 25 °C than maize
grown at 14/11 °C of 2–5 lmol m2 s1 (Naidu et al.,
2003) (Fig. 5). Even though the photosynthetic rate measured at temperatures from 10 to 25 °C in Gig-13 and
Sac-11 grown at 6/4 °C was much lower than that for
M. 9 giganteus grown at 14/11 °C (8–25 lmol m2 s1,
(Naidu et al., 2003)), this could to some extent be caused
by the increased VPD (Fig. 5), which significantly
decreased stomatal conductance (Fig. S1, A) in combination with limited Rubisco content as mentioned above.
The increase in VPD resulting in higher stomatal closure
was probably the major reason for the low A in Fest-19
during the short-term temperature increase.
Typically, the ratio between intercellular CO2 concentrations and ambient CO2 concentrations (ci/ca) is about
0.7 in C3 plants (Drake et al., 1997) and 0.3–0.4 in C4
plants (Jones, 1983). The ci/ca in Fest-19 decreased to
0.50 0.01 at 25 °C, which is significantly lower than
the typical value. With similar levels of ci/ca in Fest-19
and miscanthus genotypes at 15 °C, the C4 pathway
was more efficient in carbon fixation. The miscanthus
genotypes Gig-13 and Sac-11 had ci/ca values that were
in the 0.3–0.4 range even at 25 °C (Fig. S1, B), while the
ci/ca of Sac-12 decreased to almost 0.23 0.08 at 25 °C,
indicating that stomatal closure was limiting A in this
genotype (Fig. S1, A).
Conclusions
We found that miscanthus maintains photosynthetic
activity after growing for a longer period at 6/4 °C, and
that several genotypes increased their photosynthesis
immediately when the temperature was increased. As
expected, the photosynthetic apparatus of C3 Fest-19
was clearly more tolerant at 10/8 °C and 6/4 °C than
the C4 genotypes tested. However, also at 24/20 °C
Fest-19 was as efficient as most of the miscanthus genotypes and only Sac-12 had a significantly higher A1000
(Fig. 1a). Fest-19 may thus overall be more productive
than current miscanthus genotypes in maritime, cool,
temperate climates even though not only leaf photosynthesis but a number of other physiological factors such
as respiration and partitioning to roots are decisive for
productivity as well. We are currently growing Fest-19
and Gig-13 in side-by-side replicated field trials at two
Danish locations to obtain data on seasonal PAR interception in green leaves as well as yield data over several years. Also, we will use the leaf photosynthesis
data as well as data on portioning between plant organs
to model seasonal productivity against the harvested
yield data.
Of the miscanthus genotypes, only Sac-11 seemed to
have photosynthetic capacity at a similar high level as
Gig-13 (Hornum) in cold conditions and to recover
quickly at enhanced temperatures. Thus, Sac-11 will be
a good parent for breeding new varieties with an even
better cold tolerance than Gig-13 (Hornum) and for
broadening the genetic diversity of miscanthus if more
widespread production in cool climates is to take place.
Tin-1 did not have a high tolerance to cold conditions,
as also previously found by Jiao et al. (2016).
Acknowledgements
We thank Finn Henning Christensen for help in operating the
climate chambers and Jens Bonderup Kjeldsen for collecting
genotypes in Japan and establishing the Danish miscanthus
gene pool in 1996. The work was supported by the EU Seventh
Framework Programme (FP7/2007-2013) as part of the
GrassMargins Project (grant agreement No. 289461), and by the
Danish Council for Strategic Research as part of the BIORESOURCE Project (Grant agreement No. 11-116725).
References
Ambye-Jensen M, Johansen KS, Didion T, Kadar Z, Schmidt JE, Meyer AS (2013)
Ensiling as biological pretreatment of grass (Festulolium Hykor): The effect of
© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12342
COLD TOLERANCE OF C4 VS. C3 PLANTS 5
Temperature response curves
The temperature response of photosynthesis was measured
after seven days of growth at 6 °C in the climate chamber. The
temperature in the leaf cuvette was set at five steps: 5 °C,
10 °C, 15 °C, 20 °C and 25 °C. It was measured on three to four
replicates on the youngest fully developed leaf with each leaf
measured at all temperatures, and the CO2 concentration was
controlled at 400 ppm. For each temperature measurement,
leaves were light and temperature-acclimated until steady state
had been achieved in the gas exchange cuvette at a PAR of
1000 lmol m2 s1. Attempts were made to keep a similar leafto-air vapor pressure deficit at all temperatures by modifying
the relative humidity in the leaf cuvette. Still, an increase in
VPD could not be avoided with increasing temperatures.
The AQY values were obtained from linear regression of the
relationship between net CO2 assimilation and PAR across five
points at incident light intensities from 20 to 150 lmol m2 s1
(Long et al., 1996). A1500 was the rate of photosynthesis at a
PAR of 1500 lmol m2 s1 (A1500).
Statistical analysis
Data for each temperature treatment were analysed separately
using Analysis of Variance. If a significant effect was observed,
multiple comparisons were performed using Tukey’s HSD test.
The difference in ФPSII/ФCO2 ratio between temperature treatments for each genotype was analysed using the same methods
as mentioned above. All the analyses and tests were done in R
version 3.1.2 (Team RC, 2015). The R package ‘lsmeans’ (Lenth
& Herve, 2005) was used for multiple comparisons.
Chlorophyll fluorescence measurements
Chlorophyll fluorescence was determined on Day 6 of each
temperature settlement under the cold conditions using a MiniPAM fluorometer (Walz, Germany). The leaves were darkadapted with Dark Leaf Clips for 30 min before the measurements were performed (Bolhar-Nordenkampf et al., 1989). The
minimal fluorescence (F0) was determined at very low PPFD,
where the PSII reaction centers are in the ‘open’ state. The maximal fluorescence (Fm) was measured by applying a 0.8 s pulse
at a high light level of approx. 4000 lmol m2 s1, which forces
the closure of the reaction centers (Krause & Somersalo, 1989).
The maximum quantum yield of PSII (Fv/Fm) was calculated
from F0 and Fm, as Fv/Fm = (FmF0)/Fm. All measurements
were repeated on the six replicates of each genotype.
When an actinic light is applied on the leaf, maximum fluorescence in the light (Fm0 ) can be measured with a saturating
flash, and steady-state fluorescence (Fs) can also be recorded
just prior to the flash. The operating quantum yield of photosystem II (ΦPSII), calculated as (Fm0 Fs)/Fm0 (Genty et al.,
1989) was measured immediately after the A1000 had reached
steady state using the chlorophyll fluorescence module (CFM)
in CIRAS-2 on the ninth day after the growth temperature was
decreased. It was always measured between 8:30 and 15:00.
The instantaneous quantum cost of assimilation can be
approximated by the ratio of ΦPSII to quantum yield of CO2
assimilation (ΦCO2) (Oberhuber & Edwards, 1993). The ΦCO2
parameter is the efficiency of CO2 assimilation, which is calculated as A /(PAR*a). Leaf absorptance (a) was assumed to be
0.85 for all the genotypes in this study for the four temperature
treatments, since generally 85% of incident light can be
absorbed by leaves (Singsaas et al., 2001) and also
because apparently no dramatic changes in leaf absorptance
(approximately 0.85) take place between 450 and 700 nm for M.
9 giganteus grown at 10, 14 and 25 °C (Farage et al., 2006).
Photosynthetic response curves and calculation
Data from the light response experiment were fitted to a nonrectangular hyperbola model (Marshall & Biscoe, 1980) by
means of the nonlinear least squares curve-fitting procedure of
R for Windows (Team RC, 2015).
Results
Photosynthetic performance and operating quantum yield
of PSII
The A1000 of Sac-12 grown at 24/20 °C was 25.7 lmol
m2 s1, which was significantly higher than other genotypes except Sac-16, Sac-11 and Gig-13 (Fig. 1a). The lowest values of 18.0 and 9.8 lmol m2 s1 were measured
in Sin-15 and Tin-1, respectively, and this was significantly lower than for Sac-11, Sac-12, Gig-13, Sac-16 and
Fest-19. Concerning ΦPSII, Fest-19 and Sac-12 had significantly higher values (0.379 and 0.366, respectively) than
Sac-10, Sin-15, Gig-14 and Tin-1 (Fig. 1b).
For all genotypes, A1000 was reduced when the temperature was decreased, albeit to different extents
(Fig. 1a). At the 14/10 °C temperature, Fest-19 showed
the highest A1000 at 15.2 lmol m2 s1. This C3 species
as well as Sac-12 and Sac-11 had significantly higher
A1000 values than M. tinctorius, the two M. sinensis genotypes, Sac-17 (the Russian genotype) and Sac-16
(Fig. 1a). Fest-19 also had the highest ΦPSII at 14/10 °C.
Sin-3 and Gig-14 had significantly lower ΦPSII values
than Sac-11, Sac-12, Gig-13 and Fest-19.
When the temperature was reduced to 10/8 °C,
Fest-19 showed significantly higher A1000 (14.3 lmol
m2 s1) values than all the miscanthus genotypes of
which Sac-11, Sac-12 and Gig-13 had significantly
higher values (9.8, 8.0 and 8.9 lmol m2 s1, respectively) than all the other miscanthus genotypes
(Fig. 1a). A similar trend was observed at the 6/4 °C
conditions, where A1000 in Fest-19 was 7.5 lmol m2
s1 but 3.8 and 3.2 lmol m2 s1 in Sac-11 and Gig13, respectively, which is significantly higher than for
the other miscanthus genotypes except for Sac-12 and
Sin-15. The highest ΦPSII value was observed in Fest19 at both 10/8 °C and 6/4 °C (Fig. 1b). Within the
© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12342
6 X . J I A O et al.
Fig. 1 (a) Net photosynthetic rate at 1000 lmol m2 s1 (A1000), (b) Quantum yield of photosystem II (ΦPSII) at 1000 lmol m2 s1,
(c) The ratio of operating efficiency of PSII to apparent quantum yield of CO2 assimilation (ΦPSII/CO2) under warm day/night
(24/20 °C) and three cold (14/10 °C, 10/8 °C and 6/4 °C) growing conditions. Error bars represent SE (n = 6). Values without letters
in common are significantly different at the P = 0.05 level within each temperature treatment.
miscanthus genotypes, Sac-11, Sac-12 and Gig-13
showed significantly higher ΦPSII values (0.191, 0.157
and 0.195, respectively) than other genotypes at 10/
8 °C. There was no significant difference in ΦPSII
values between the miscanthus genotypes at 6/4 °C
(Fig. 1b).
The ΦPSII/ΦCO2 ratio
The ratio ΦPSII/ΦCO2 increased after temperature
reduction for the miscanthus genotypes. However, there
was no significant change in Fest-19 when the temperature was decreased to 14/10 °C, 10/8 °C and even
© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12342
COLD TOLERANCE OF C4 VS. C3 PLANTS 7
6/4 °C. The ΦPSII/ΦCO2 ratio for Tin-1 and Fest-19
was significantly higher than for the other miscanthus
genotypes at 24/20 °C (Fig. 1c). At 14/10 °C, Sac-12,
Sac-11 and Gig-13 had the lowest values compared to
the other genotypes, but they were not significantly different from other genotypes except Tin-1. When the
temperature decreased from 14/10 °C to 10/8 °C a significant increase was found in Sac-11, Sac-12, Gig-13,
Gig-14 and Sac-17. At 10/8 °C, Sin-3, Gig-13, Sac-11and
Sac-12 had significantly lower values than all other
genotypes except Sin-15 and Sac-17 (Fig. 1c). The
ΦPSII/ΦCO2 ratio in Sin-3, Sac-11 and Sin-15 increased
significantly from 10/8 °C to 6/4 °C, whereas for Sac-18
it decreased (not significantly). At 6/4 °C, the ratio was
significantly higher in Sin-3, Tin-1 and Sac-16 than in
Sac-12, Gig-13, Sac-18 and Fest-19.
Chlorophyll fluorescence
The C3 crop Fest-19 had an Fv/Fm value of 0.82 when it
was grown at 24/20 °C, while in the miscanthus genotypes values were between 0.73 and 0.80 (Fig. 2). Three
genotypes, Sac-10, Sac-11 and Sac-16, had significantly
higher Fv/Fm values of 0.80, 0.79, and 0.80, respectively,
than the 0.74 and 0.73, respectively, of Gig-14 and Tin-1
at 24/20 °C.
A reduction in Fv/Fm was observed in all miscanthus
genotypes during the temperature decrease, but was
less clear for Fest-19 (Fig. 2). Tin-1 and Gig-14 had
significantly lower values than other genotypes at 14/
10 °C. When the temperature was further decreased to
10/8 °C, there was a slight decrease in Fest-19, but it
was much greater in miscanthus. The Fv/Fm values of
0.68, 0.65 and 0.67, respectively, for Gig-13, Sac-11 and
Sac-12 were significantly higher than the 0.43, 0.44, 0.51,
0.48 and 0.51 for Sin-3, Tin-1, Sac-16, Gig-14 and Sac-18.
At 6/4 °C, the Fv/Fm value in Fest-19 Fv/Fm was 0.71.
This was significantly higher than all the miscanthus
genotypes. At this temperature there was no significant
difference between Gig-13, Sac-11, Sac-12 and Sin-15,
but they had significantly higher values than Sin-3, Sac10, Sac-16 and Gig-14 (Fig. 2). For both Fest-19 and the
miscanthus genotypes, the largest reduction was
observed when the temperature dropped from 10/8 °C
to 6/4 °C.
Light response curves at different temperature levels
The responses of CO2 assimilation to light showed significant effects of temperature on genotypes at each
measured light level (Fig. 3). Sac-12 and Fest-19 showed
significantly higher AQY (0.043 and 0.041) than other
measured genotypes at 24/20 °C (Fig. 4). A significant
reduction of AQY was observed in the measured genotypes when the temperature was decreased from 24/
20 °C to 14/10 °C. However, there was no significant
reduction of AQY for Sac-11, Sac-12, Gig-13 and Fest-19
when the temperature was further decreased from 14/
10 °C to 10/8 °C, while Tin-1 and Sin-15 had a decrease
of 38% and 52%, respectively. No significant difference
in AQY was observed between Sac-11, Sac-12, Gig-13
and Fest-19 grown at 14/10 °C and 10/8 °C. At 6/4 °C,
Fest-19 maintained the same AQY level as when it was
grown under 14/10 °C, while the miscanthus genotypes
Fig. 2 Maximum quantum yield of PSII (Fv/Fm) measured under warm day/night (24/20 °C) and three cold (14/10 °C, 10/8 °C
and 6/4 °C) growing conditions. Error bars represent SE (n = 6). Values without letters in common are significantly different at the
P = 0.05 level within each temperature treatment.
© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12342
8 X . J I A O et al.
experienced a significant reduction when the temperature fell from 10/8 °C to 6/4 °C.
Sac-12 had the highest A1500 value of 30.6 lmol m2
1
s at 24/20 °C (Fig. 4b). This was significantly higher
than for Fest-19 (25.1 lmol m2 s1) and the other genotypes, except Gig-13 (27.9 lmol m2 s1). However, Fest19 maintained the highest A1500 compared to the miscanthus genotypes at the three cold temperatures. Of the
miscanthus genotypes at 14/10 °C, Sac-11 had a significantly higher A1500 (14.8 lmol m2 s1) than Sin-15 and
Tin-1 (11.1 and 6.1 lmol m2 s1, respectively). When
the temperature was reduced to 10/8 °C, Sac-11 as well
as Sac-12 and Gig-13 had significantly higher A1500
values than Sin-15 and Tin-1. Furthermore, Sac-11 was
significantly higher than Sin-15 and Tin-1 at 6/4 °C.
Short-term temperature response curves at 6/4 °C
growing temperature
Gig-13 and Sac-11 showed a significant increase in A1000
when the temperature was increased from 5 °C to
25 °C, while Sac-12 only increased when the temperature rose from 5 °C to 10 °C and remained steady during further temperature increases (Fig. 5). However, the
A1000 of Fest-19, which showed the highest level at 5 °C,
decreased significantly when the temperature was
increased to 15 °C, as the leaf-to-air vapor pressure deficit increased during the temperature increase (from 0.4
to 2.5 kPa), inducing stomatal closure (Fig. S1, A).
Discussion
Photosynthetic performance at 14 °C
The constraint to C4 photosynthesis in miscanthus in
cold conditions might differ depending on the temperature. We observed a decrease in A1000 and ΦPSII in all
the miscanthus genotypes and in festulolium for each
temperature reduction. However, M. 9 giganteus has
reportedly markedly higher photosynthetic rates at
14 °C compared to other C4 species like maize (Naidu
& Long, 2004; Wang et al., 2008b) and Cyperus longus L.
(Farage et al., 2006). In the present study, Sac-11, Sac-12
and Gig-13 maintained similar photosynthetic rates to
the C3 grass Fest-19 at 14 °C (Fig. 1a). This indicates
that some genotypes of miscanthus might be useful for
cultivation in maritime temperate conditions. Our previous study suggests that a reduced photosynthetic activity in miscanthus due to a temperature decrease to
14 °C could be reversed in these cold-tolerant genotypes
once the temperature is increased again (Jiao et al.,
2016). Another study shows that the effect of a low
growth temperature (14 °C) on photosynthesis in the C4
grass M. glomerata could be reversed by a 24-h exposure
to higher temperatures (Kubien & Sage, 2004a). This
suggests that such a reversible photoinhibition at 14 °C
is lacking in maize (Naidu et al., 2003), but is a strategy
for some C4 species to tolerate cool climates (Kubien &
Sage, 2004a).
Fig. 3 Light response curves (net photosynthesis, An, vs. photosynthetic active radiation, PAR) of selected miscanthus genotypes
and festulolium under warm day/night (24/20 °C) and three cold (14/10 °C, 10/8 °C and 6/4 °C) growing conditions. Each point is
the fitted mean value from a nonrectangular hyperbolic model of six measurements. Tin-1 was not included in 6/4 °C.
© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12342
COLD TOLERANCE OF C4 VS. C3 PLANTS 9
Fig. 5 Net photosynthetic CO2 uptake at a PAR of
1000 lmol m2 s1 vs. temperature for Sac-11, Sac-12, Gig-13
and Fest-19 grown at 6/4 °C (day/night) for 7 days, and the
change of leaf-to-air vapor pressure deficit with temperature.
Error bars represent SE (n = 3 or 4).
Fig. 4 (a) Apparent quantum yield of CO2 uptake (AQY), and
(b) Net photosynthetic rate at a PAR of 1500 lmol m2 s1
(A1500). The parameters are measured on five miscanthus
genotypes and festulolium under warm day/night (24/20 °C)
and three cold growing conditions (14/10 °C, 10/8 °C and 6/
4 °C). Error bars represent SE (n = 6). Values without letters in
common are significantly different at the P = 0.05 level within
each temperature treatment.
Generally, C4 plants show their photosynthetic advantage over C3 plants at temperatures above 20 °C
(Yamori et al., 2014). However, here we find that some
miscanthus genotypes can compete with C3 festulolium
at a temperature of 14 °C.
For C4 plants there is linear relationship between
ΦPSII and ΦCO2 under nonstressed conditions over a
range of light intensities (Genty et al., 1989; Krall &
Edwards, 1990). No significant increase in ΦPSII/ΦCO2
was found for any of the genotypes in this study when
the temperature was lowered from 24/20 °C to14/10 °C
(Fig. 1c). This is consistent with previous findings that
no significant changes happen to the ΦPSII/ΦCO2 ratio
in M. 9 giganteus or Z. mays leaves when the temperature falls from 25/20 °C to 14/11 °C (Naidu &
Long, 2004). This indicates that the electron transport
through PSII is just sufficient for the CO2 assimilation
requirement, and that no alternative electron sinks exist
at 14 °C for any of the miscanthus genotypes tested in
this study.
Another interesting parameter is the ratio of Fv/Fm,
which gives a relative measure of the maximum quantum efficiency of PSII photochemistry (Genty et al.,
1989). Decreases in Fv/Fm can be due to the development of nonphotochemical quenching processes or
photo damage to PSII reaction centers (Baker & Rosenqvist, 2004). We observed no decrease in Fv/Fm for
Fest-19 at 14 °C, but a slight decrease (within 10%) compared with the values measured at 24 °C was detected
in Sac-11, Sac-12 and Gig-13 (Fig. 2). This suggests that
a temperature of 14 °C had no damaging effect on the
photosynthetic apparatus in the C3 grass, but that a
minor low-temperature-induced photo inhibition
occurred in the three genotypes of miscanthus, even
though the A1000 values for the other tested genotypes,
except Sac-10, were significantly lower than the three
apparently cold-tolerant genotypes (Fig. 1a). Interestingly, these other genotypes also had a significantly
lower ΦPSII (Fig. 1b), which means that markedly
less light absorbed by PSII antennae is used for photochemistry. Reduced A1000 could be the direct result of
reduced PSII efficiency due to PSII photo inhibition.
The largest decrease in A1000 when the temperature
was lowered from 24/20 °C to 14/10 °C occurred in
Sac-16. The plants of this genotype turned reddish violet
when they were transferred from 15 °C in the greenhouse to the 24/20 °C climate chamber conditions. This
could be due to anthocyanin synthesis during temperature changes (Pietrini & Massacci, 1998). Like Sin-3 and
Sin-15, the Sac-17 genotype suffered a large reduction in
A1000 after the temperature decrease to 14/10 °C and
© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12342
10 X . J I A O et al.
turned yellow after the repositioning from the greenhouse to the climate chamber, i.e. there was a visible
chlorophyll reduction. These two genotypes thus
seemed to be stressed both by the warm temperatures
and by subsequent cold conditions.
Photosynthetic performance at 10 and 6 °C
When the temperature was lowered to 10/8 °C, further
decreases in A1000 and Fv/Fm values were found in
miscanthus but not in festulolium (Fig. 2), which indicates that additional increases in nonphotochemical
quenching and/or photo damage of the PS II reaction
center occurred in miscanthus. One of the major
mechanisms for miscanthus to get rid of excess
energy absorbed by the leaves is heat dissipation. For
M. 9 giganteus grown at 10 °C high levels of nonphotochemical quenching of excitation energy has been associated with a 20-fold increase in zeaxanthin content in
dark-adapted leaves, as a photo-protective mechanism
(Farage et al., 2006). Sac-11, Sac-12 and Gig-13 showed
the same AQY values at 10/8 °C as at 14/10 °C
(Fig. 4a), which further indicates that no photo damage
happened to these three genotypes when the temperature was decreased from 14/10 °C to 10/8 °C. The significantly higher AQY values and A1500 in the three
genotypes obviously would make them more suitable
for cold climates than Tin-1 and Sin-15 at 10/8 °C
(Fig. 4), due to their significantly higher CO2 uptake
under both light-limited and light-saturated conditions.
No significant decrease was observed in A1000 in Fest-19
during the temperature reduction from 14/10 °C to 10/
8 °C; the relatively high ΦPSII and Fv/Fm in Fest-19
might have played a major role but it was not the only
possible reason for maintaining the high photosynthetic
rate. An alternative explanation might be the Rubisco
content since C3 species generally have three to six
times as much Rubisco as C4 species (Ku et al., 1979).
Interestingly, all the miscanthus genotypes continued
to display photosynthetic activity at 6/4 °C, though
A1000 was quite low for several of them (Fig. 1a). The
values of A1000 in Gig-13 grown for 6 days at, respectively, 10/8 °C and 6/4 °C were 8.9 and 3.2 lmol m2
s1, which is similar to previously reported values for
M. 9 giganteus where the photosynthetic rate was measured during short-term exposure to low temperatures.
Measured values in that study were approximately
9 lmol m2 s1 at 10 °C and 4 lmol m2 s1 at 5 °C for
plants that had previously been grown at 14/11 °C and
25/14 °C, respectively (Naidu et al., 2003). Thus, longterm exposure to cold temperatures down to 6 °C in M.
9 giganteus did not have a detrimental effect on the
photosynthetic apparatus. This suggests that the
decrease in photosynthetic capacity in Gig-13 and
Sac-11 at low temperatures (above 5 °C) could be
reversed if the temperature was raised again, and our
result of the temperature response curve also supported
this (Fig. 5). This could be one of the reasons for the
remarkable capacity of M. 9 giganteus to grow in cool
climates and produce a much higher yield compared
with another C4 plant, Spartina cynosuroides, in the
United Kingdom (Beale & Long, 1995). An earlier miscanthus growth model, MISCANMOD, uses a base temperature of 10 °C, which was considered as the starting
temperature for leaf growth (Hastings et al., 2009). Our
results suggest that the base temperature for miscanthus
could be lower than 6 °C.
The reduction in AQY in Sac-11, Sac-12 and Gig-13
from 10/8 °C to 6/4 °C might be because of a further
accumulation of zeaxanthin even at the low light intensities, which reduce the light-harvesting efficiency
under light-limiting conditions (Long et al., 1994; Fryer
et al., 1995). However, this phenomenon was not
observed in Fest-19, which showed no significant
changes in AQY from 14/10 °C to 6/4 °C (Fig. 4a). This
significantly higher tolerance to the cold in Fest-19 was
also observed at 2 °C. A1000 for Fest-19 grown at 6/4 °C
and measured at 2 °C was 5.72 lmol m2 s1 (data not
shown), which was similar to data measured for Lolium
perenne L. grown and measured at 2 °C (Hoglind et al.,
2011).
The significantly higher AQY and A1500 in Fest-19
than in the miscanthus genotypes even at 6/4 °C
(Fig. 4a) and high A1000 at 2 °C can support the early
emergence and longer growing season of Fest-19. The
daily mean temperature in Denmark in 2013 and 2014
was around 8.7 °C, ranging from -8.6 to 23 °C, and
there were about 200 days each year with a mean temperature below 10 °C (data collected from climate station in Foulumgaard, Denmark, 56°300 N, 9°350 E). Thus,
the significantly higher photosynthesis in Fest-19 in the
2–10 °C temperature range can be the reason for its high
biomass production when grown in temperate
conditions. Yields in Denmark of more than 20 T DM
ha1 for this cultivar have been recorded (Manevski
et al., 2015).
However, we observed a tendency of an increasing
ΦPSII/ΦCO2 ratio when lowering the temperature from
14/10 °C to 6/4 °C for all the miscanthus genotypes,
but not for Fest-19. This indicates that the quantum efficiency of the electron flux through PSII relative to the
quantum efficiency of CO2 assimilation in miscanthus
increased as the temperature decreased, due to additional electron sinks. It has been suggested that elevated
ΦPSII/ΦCO2 ratio in mature maize leaves under cold
conditions is due to O2 being used as an alternative
electron accepter (Fryer et al., 1998), which is also
a mechanism for preventing photo damage to the
© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12342
C O L D T O L E R A N C E O F C 4 V S . C 3 P L A N T S 11
photosynthetic apparatus (Farage et al., 2006). A previous study also found an increased ФPSII/ФCO2 ratio
for M. 9 giganteus grown at 10 °C compared with 24 °C
and 14 °C (Farage et al., 2006). Photoinhibitory damage
has been found to be a major challenge for C4 plants
grown at cold temperatures, for example, maize grown
at 17 °C or below (Long et al., 1983). It seems that M. 9
giganteus has the ability to minimize this damage by a
massive increase in zeaxanthin content to facilitate heat
dissipation in conjunction with induction of electron
transport to an acceptor other than CO2 (Farage et al.,
2006).
However, CO2 leakiness from the bundle sheath cells
back to mesophyll is an alternative energy expenditure
in the C4 photosynthetic pathway under high light conditions. Especially at low temperatures, where the
Rubisco capacity is an important limitation for sustaining C4 photosynthesis (Sage, 2002; Kubien et al., 2003),
the ΦPSII/ΦCO2 ratio will increase (Von Caemmerer,
2000). Increased values of ΦPSII /ΦCO2 at cool temperatures have also been observed in Muhlenbergia glomerata
measured at 10 and 5 °C by Kubien & Sage (2004b),
who concluded that leaking of CO2 through the bundle
sheaths was increased due to limited Rubisco capacity
(Rubisco content) at low temperatures. The significantly
higher ΦPSII /ΦCO2 ratio at 6/4 °C in our study might
indicate that Rubisco posed a major limitation to photosynthesis in all the miscanthus genotypes but not in
Fest-19. However, the underlying reasons for the
increase of ΦPSII /ΦCO2 were not determined in this
study.
Gig-13 and Sac-11 grown at 6/4 °C in the present
study showed much higher rates of photosynthesis at
all measured temperatures from 5 to 25 °C than maize
grown at 14/11 °C of 2–5 lmol m2 s1 (Naidu et al.,
2003) (Fig. 5). Even though the photosynthetic rate measured at temperatures from 10 to 25 °C in Gig-13 and
Sac-11 grown at 6/4 °C was much lower than that for
M. 9 giganteus grown at 14/11 °C (8–25 lmol m2 s1,
(Naidu et al., 2003)), this could to some extent be caused
by the increased VPD (Fig. 5), which significantly
decreased stomatal conductance (Fig. S1, A) in combination with limited Rubisco content as mentioned above.
The increase in VPD resulting in higher stomatal closure
was probably the major reason for the low A in Fest-19
during the short-term temperature increase.
Typically, the ratio between intercellular CO2 concentrations and ambient CO2 concentrations (ci/ca) is about
0.7 in C3 plants (Drake et al., 1997) and 0.3–0.4 in C4
plants (Jones, 1983). The ci/ca in Fest-19 decreased to
0.50 0.01 at 25 °C, which is significantly lower than
the typical value. With similar levels of ci/ca in Fest-19
and miscanthus genotypes at 15 °C, the C4 pathway
was more efficient in carbon fixation. The miscanthus
genotypes Gig-13 and Sac-11 had ci/ca values that were
in the 0.3–0.4 range even at 25 °C (Fig. S1, B), while the
ci/ca of Sac-12 decreased to almost 0.23 0.08 at 25 °C,
indicating that stomatal closure was limiting A in this
genotype (Fig. S1, A).
Conclusions
We found that miscanthus maintains photosynthetic
activity after growing for a longer period at 6/4 °C, and
that several genotypes increased their photosynthesis
immediately when the temperature was increased. As
expected, the photosynthetic apparatus of C3 Fest-19
was clearly more tolerant at 10/8 °C and 6/4 °C than
the C4 genotypes tested. However, also at 24/20 °C
Fest-19 was as efficient as most of the miscanthus genotypes and only Sac-12 had a significantly higher A1000
(Fig. 1a). Fest-19 may thus overall be more productive
than current miscanthus genotypes in maritime, cool,
temperate climates even though not only leaf photosynthesis but a number of other physiological factors such
as respiration and partitioning to roots are decisive for
productivity as well. We are currently growing Fest-19
and Gig-13 in side-by-side replicated field trials at two
Danish locations to obtain data on seasonal PAR interception in green leaves as well as yield data over several years. Also, we will use the leaf photosynthesis
data as well as data on portioning between plant organs
to model seasonal productivity against the harvested
yield data.
Of the miscanthus genotypes, only Sac-11 seemed to
have photosynthetic capacity at a similar high level as
Gig-13 (Hornum) in cold conditions and to recover
quickly at enhanced temperatures. Thus, Sac-11 will be
a good parent for breeding new varieties with an even
better cold tolerance than Gig-13 (Hornum) and for
broadening the genetic diversity of miscanthus if more
widespread production in cool climates is to take place.
Tin-1 did not have a high tolerance to cold conditions,
as also previously found by Jiao et al. (2016).
Acknowledgements
We thank Finn Henning Christensen for help in operating the
climate chambers and Jens Bonderup Kjeldsen for collecting
genotypes in Japan and establishing the Danish miscanthus
gene pool in 1996. The work was supported by the EU Seventh
Framework Programme (FP7/2007-2013) as part of the
GrassMargins Project (grant agreement No. 289461), and by the
Danish Council for Strategic Research as part of the BIORESOURCE Project (Grant agreement No. 11-116725).
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Additional Supporting Information may be found in the
online version of this article:
Figure S1 (a) stomatal conductance (Gs), and (b) the ratio
between intercellular CO2 concentrations and ambient CO2
concentrations (ci/ca) vs. temperature for Sac-11, Sac-12,
Gig-13 and Fest-19 grown at 6/4 °C (day/night), Error bars
represent SE (n = 3 or 4).
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© 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12342